Primary Microglia Isolation from Postnatal Mouse Brains
Summary
Primary cell culture is one of the primarily used approaches for studying microglial biology in vitro. Here, we developed a method for simple and rapid microglia isolation from the mouse postnatal day 1 (P1) to P4.
Abstract
Microglia are the mononuclear phagocytes in the central nervous system (CNS), which play key roles in maintaining homeostasis and regulating the inflammatory process in the CNS. To study the microglial biology in vitro, primary microglia show great advantages compared to immortalized microglial cell lines. However, microglia isolation from the postnatal mouse brain is relatively less efficient and time-consuming. In this protocol, we provide a quick and easy-to-follow method to isolate primary microglia from the neonatal mouse brain. The overall steps of this protocol include brain dissection, primary brain cell culture, and microglia isolation. Using this approach, researchers can obtain primary microglia with high purity. In addition, the harvested primary microglia were able to respond to the lipopolysaccharides challenge, indicating they retained their immune function. Collectively, we developed a simplified approach to efficiently isolate primary microglia with high purity, which facilitates a wide range of microglial biology investigations in vitro.
Introduction
Microglia, the resident immune cells in the central nervous system (CNS), play pivotal roles in the maintenance of homeostasis, which respond to the neuropathological challenges1. Recently, intensive investigations have been conducted to figure out the physiological functions of microglia, e.g., in Alzheimer's disease2. Currently, the transcriptional profile of microglia at the single-cell resolution obtained during the CNS development, aging, and disease provides a better understanding of microglial function in healthy and diseased brains3. Previous studies identified a disease-associated microglial subtype in AD and other neurodegenerative diseases4,5,6. This sub-population is proximal to the amyloid β (Aβ) deposition. Genes associated with phagocytosis and lipid metabolism (e.g., Apoe, Tyrobp) were found to be up-regulated in these populations6,7. However, the subcellular processes, extracellular and intracellular signaling pathways that regulate the dynamic molecular profile changes in microglia are not fully understood. Particularly, the mechanism underlying chronic microglial activation in neurodegenerative diseases remains elusive. Therefore, it is crucial to understand microglia-involved cellular mechanisms since microglia responses clearly contribute to brain development and the progression of neurodegeneration.
Although there are a couple of in vivo and in vitro tools to study microglia, there are still some limitations. It is technically difficult to obtain a large enough number of microglial cells with high purity for routinely carrying out experiments, such as western blot, to elucidate intracellular signaling pathways. Primary microglia culture provides an alternative means to study the biology of microglia. The cultured primary microglia can be applied to analyze microglial phagocytic capacity after gene manipulation, and evaluate pro- and anti-inflammatory cytokines production in response to inflammatory stimuli, and other biological aspects to understand their roles in the brain8. Here, we present a new protocol and describe a step-by-step instruction for the isolation and culture of primary microglia from neonatal mouse brain, with emphasis on the steps that are critical for obtaining healthy and pure primary microglial cells.
Protocol
Representative Results
Discussion
This protocol is based on the methods described previously with some modifications11. Tips for improving the viability and purity of isolated microglia are listed as follows. First, take care to avoid contamination when preparing buffers used for tissue isolation and cell culture. Make sure the surgery tools, containers, and plastic equipment are sterile. Usually, we perform the brain dissection in a separate tissue culture hood aside from the cell culture hood for general cell culture to avoid cr…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to show their respects to the heroes combating the COVID-19 outbreak. The authors thank Fang Lei (Fudan University) for the excellent laboratory management, Dr. Zikai Zhou, Dr. Jing Li, and Dr. Guiqing He (Shanghai Mental Health Center) for the discussion of microglia isolation. Last but not least, the authors show their gratitude and respect to all animals sacrificed in this study. This study was supported by the National Key R&D Program of China (Grant No. 2017YFC0111202) (B.P.), National Natural Science Foundation of China (Grant No. 31922027) (B.P.) and (Grant No. 32000678) (Y.R.), and Shenzhen Science and Technology Research Program (Grant No. JCYJ20180507182033219 and JCYJ20170818163320865) (B.P.) and (Grant No. JCYJ20170818161734072) (S.X.).
Materials
| Cell strainers, 40 µm | ThermoFisher Scientific | 22-363-547 | |
| DNase I | Sigma | 11284932001 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco by Life Technologies | C11995500BT | |
| Dulbecco's Phosphate Buffered Saline (DPBS) | Gibco by Life Technologies | 14190-144 | |
| Fetal Bovine Serum (FBS) | Gibco by Life Technologies | 10099141 | |
| Papain, Suspension | Sangon Biotech | Papain, Suspension | |
| Penicillin-Streptomycin 100X solution | Hyclone | SV30010 | |
| Poly-D-Lysine | ThermoFisher Scientific | A3890401 |
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